Omega-3 fatty acid desaturase

ABSTRACT

Recombinant expression of fat-1 gene of  Caenorhabditis elegans  in a wide variety of cells, including cells of  Arabidopsis thaliana  and  Saccharomyces cerevisiae,  produces a polypeptide having ω-3 desaturase activity.

CROSS REFERENCE TO RELATED CASE

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/038,409, filed Feb. 18, 1997, which is incorporatedherein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under ResearchGrant 95-37301-2287 from the USDA-NRICGP. The Government has certainrights to this invention.

BACKGROUND OF THE INVENTION

[0003] This invention relates to fatty acid metabolism, in particular tofatty acid desaturases.

[0004] Polyunsaturated fatty acids are important as structuralcomponents of membrane glycerolipids and as precursors of families ofsignaling molecules including prostaglandins, thromboxanes, andleukotrienes (Needleman et al. , Annu. Rev. Biochem. 55:69-102, 1986;Smith and Borgeat, in Biochemistry of Lipids and Membranes, eds. Vanceand Vance, Benjamin/Cummings, Menlo Park, Calif., 1986, pp. 325-360).

[0005] The principal fatty acid precursors of these signaling moleculesare arachidonic acid (Δ5,8,11, 14-20:4), providing an ω-6 substrate thatis responsible for the major synthesis of these compounds, andeicosapentanenoic acid (Δ5,8,11,14,17-20:5), an ω-3 substrate that isresponsible for the parallel synthesis of many elcosanoids having anadditional double bond. An important class of enzymes involved in thesynthesis of polyunsaturated fatty acids is the fatty acid desaturases,which catalyze the introduction of double bonds into the hydrocarbonchain.

[0006] In vertebrates, desaturases are known to act at the Δ4, 5, 6, 8and 9 positions (Holloway, In: The Enzymes, ed. Boyer, Academic Press,New York, vol. 16, 1983, pp. 63-83). The 18:0-CoA Δ9 desaturase from ratliver has been characterized biochemically (Strittmatter et al., Proc.Natl. Acad. Sci. USA 71:4565-4569, 1974; Thiede et al., J. Biol. Chem.260:14459-14463, 1985), and the corresponding gene has been cloned(Thiede et al., J. Biol. Chem. 261:13230-13235, 1986). However, theremaining four enzymes have remained recalcitrant to purification andgenes that encode them have not been isolated. Based on availableinformation, and by analogy to the 18:0-CoA desaturase, it is likelythat the remaining four enzymes are integral membrane proteins thatrequire other membrane components (cytochrome b₅ and NADH:cytochrome b₅reductase) for activity (Strittmatter et al., Proc. Natl. Acad. Sci. USA71:4565-4569, 1974), and it is these features that have limited progressin studying the biochemistry and molecular genetics of these importantsynthetic reactions.

[0007] Biochemical studies of membrane-bound fatty acid desaturases inplants have proven equally difficult, and only one enzyme has beenpurified to homogeneity (Schmidt et al., Plant Mol. Biol. 26:631-642,1994). Higher plants produce many different unsaturated fatty acids(Hilditch and Williams, The Chemical Constituents of Natural Fats,Chapman and Hall, London, 4th Ed., 1964), but in membrane lipids themajor locations for double bonds are at the Δ9, 12 and 15 (ω-3)positions of 18-carbon acyl chains and the corresponding Δ7, 10 and 13(ω-3) positions of 16-carbon chains (Browse and Somerville, Ann. Rev.Plant Physiol. Plant Mol. Biol. 42:467-5069, 1991).

SUMMARY OF THE INVENTION

[0008] According to one embodiment of the invention, a cell is providedthat includes a recombinant FAT-1 polypeptide that desaturates an ω-6fatty acid of the cell to a corresponding ω-3 fatty acid. FAT-1 iscapable of desaturating ω-6 fatty acids having carbon chains of at least18 carbons (e.g., 20-to 22-carbon fatty acids), and is significantlymore efficient than FAD3, for example, at desaturating ω-6 fatty acidshaving carbon chains of 20 carbons or longer, producing lipids having atleast 25% of 20-carbon ω-6 fatty acids desaturated to the correspondingω-3 fatty acid. FAT-1 can desaturate double bonds at positions Δ4, Δ5,Δ6, Δ7, and Δ8, for example. The expression of the FAT-1 polypeptide ina cell permits the cell to have a greater proportion of the ω-3 fattyacid than an otherwise similar cell lacking the FAT-1 polypeptide,including cells from a wide variety of organisms, such as bacteria,cyanobacteria, phytoplankton, algae, fungi, plants, and animals.

[0009] According to another aspect of the invention, the recombinantFAT-1 polypeptide has at least 60% amino acid sequence identity with theFAT-1 polypeptide shown in FIG. 1 (SEQ ID NO: 1 and 2). In preferredembodiments, the recombinant FAT-1 polypeptide has only conservativeamino acid substitutions to the FAT-1 polypeptide of FIG. 1.

[0010] According to another aspect of the invention, the recombinantFAT-1 polypeptide is encoded by a polynucleotide that includes asequence having at least 70% nucleotide sequence identity with the fat-1polynucleotide sequence of FIG. 1. For example, according to oneembodiment, such a polynucleotide includes a full-length native fat-1protein-coding region, e.g., the protein-coding region of the fat-1polynucleotide sequence of FIG. 1.

[0011] According to another aspect of the invention, lipids are providedthat are produced from such cells.

[0012] According to another aspect of the invention, transgenic plantsare provided that include a fat-1 polynucleotide that is expressible inat least a part of the plant, e.g., in seeds of the plant. Also providedare seeds of such transgenic plants. Also provided are lipids from suchtransgenic plants that have higher proportions of ω-3 fatty acids thancontrol lipids obtained from otherwise similar plants lacking the fat-1polynucleotide.

[0013] According to another aspect of the invention, related methods ofdesaturating an ω-6 fatty acid to a corresponding ω-3 fatty acid areprovided. Such methods comprise the steps of: (a) providing a cell thatcomprises a recombinant FAT-1 polypeptide; and (b) growing the cellunder conditions under which the FAT-1 polypeptide desaturates an ω-6fatty acid to produce a corresponding ω-3 fatty acid.

[0014] According to another aspect of the invention, related methods ofproducing a lipid comprising an ω-3 fatty acid are provided that includethe steps of: (a) providing a lipid that includes an ω-6 fatty acid; and(b) desaturating at least some of the ω-6 fatty acid to a correspondingω-3 fatty acid with a recombinant FAT-1 polypeptide. For example, such amethod can be practiced by expressing a recombinant fat-1 nucleic acidin a cell, thereby producing a recombinant FAT-1 polypeptide in thecell.

[0015] The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the nucleotide sequence of the fat-1 cDNA and thededuced amino-acid sequence of the FAT-1 polypeptide encoded by thecDNA.

DETAILED DESCRIPTION OF THE INVENTION

[0017] We have cloned fat-1, an ω-3 fatty acyl desaturase gene fromCaenorhabditis elegans. When expressed in a wide range of host cells,the polypeptide encoded by fat-1 catalyzes the introduction of an ω-3double bond into 18-, 20-, and 22-carbon fatty acids.

[0018] The C. elegans fat-1 gene encodes the first animal representativeof a class of glycerolipid desaturases that have previously beencharacterized in plants and cyanobacteria. The FAT-1 protein is an ω-3fatty acyl desaturase that recognizes a range of 18-, 20-, and 22-carbonω-6 substrates. When expressed in a wide range of host cells, FAT-1catalyzes the introduction of an ω-3 double bond into 18-, 20-, and22-carbon fatty acids. The efficiency of FAT-1 in desaturating 20- and22-carbon substrates appears to be much greater than FAD3 desaturase ofArabidopsis, for example.

[0019] A recombinant ω-3 fatty acyl desaturase polypeptide, e.g., aFAT-1 polypeptide, is useful for producing lipids having a higherproportion of ω-3 fatty acids, whether by means of recombinantexpression in a cell or in an industrial processes using purified FAT-1polypeptide. Such lipids are useful as food oils, as nutritionalsupplements, and as chemical feedstocks, for example.

[0020] Definitions and Methods

[0021] The following definitions and methods are provided to betterdefine the present invention and to guide those of ordinary skill in theart in the practice of the present invention. Unless otherwise noted,terms are to be understood according to conventional usage by those ofordinary skill in the relevant art. Definitions of common terms inmolecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

[0022] Nucleic Acids

[0023] “Polynucleotide”. A polynucleotide (or nucleic acid) sequence isa naturally-occurring or chemically-synthesized DNA or RNA sequence. Apolynucleotide according to the invention may be single- ordouble-stranded.

[0024] “fat-1 Polynucleotide”; “fat-1 Gene”. The terms “fat-1polynucleotide” or “fat-1 gene” refer to a native FAT-1-encodingpolynucleotide or a fragment thereof, e.g., a native C. elegans cDNA orgenomic sequence or alleles, or fat-1 homologs from other species. Theterms also encompass variant forms of a native fat-1 polynucleotidesequence or fragment thereof as discussed below, includingpolynucleotides that encodes a polypeptide having FAT-1 biologicalactivity.

[0025] Native fat-1 sequences can include 5′- and 3′-flanking sequencesor internal sequences operably linked to a native fat-1 polynucleotidesequence, including regulatory elements and/or intron sequences.

[0026] “FAT-1 Biological Activity”. The term “FAT-1 biological activity”refers to a biological activity characteristic of a native FAT-1polypeptide.

[0027] “Native”. The term “native” refers to a naturally-occurring(“wild-type”) polynucleotide or polypeptide.

[0028] “Homolog”. A “homolog” of fat-1 is a polynucleotide from aspecies other than C. elegans that encodes a polypeptide that isfunctionally similar to FAT-1 and that preferably has at least 60%amino-acid sequence similarity, or more preferably, at least 60%sequence identity, to FAT-1.

[0029] “Isolated”. An “isolated” polynucleotide is one that has beensubstantially separated or purified away from other polynucleotidesequences in the cell of the organism in which the polynucleotidenaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA, by conventional nucleic acid-purification methods. The term alsoembraces recombinant polynucleotides and chemically synthesizedpolynucleotides.

[0030] Fragments, Probes, and Primers. A fragment of a fat-1polynucleotide is a portion of a fat-1 polynucleotide that is less thanfull-length and comprises at least a minimum length capable ofhybridizing specifically with a native fat-1 polynucleotide understringent hybridization conditions. The length of such a fragment ispreferably at least 15 nucleotides, more preferably at least 20nucleotides, and most preferably at least 30 nucleotides of a nativefat-1 polynucleotide.

[0031] Polynucleotide probes and primers can be prepared based on anative fat-1 polynucleotide. A “probe” is an isolated polynucleotide towhich is attached a conventional detectable label or reporter molecule,e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme.A “primer” is an isolated polynucleotide that can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target polynucleotide strand, thenextended along the target polynucleotide strand by a polymerase, e.g., aDNA polymerase. Primer pairs can be used for amplification of apolynucleotide sequence, e.g., by the polymerase chain reaction (PCR) orother conventional amplification methods.

[0032] Probes and primers are generally 15 nucleotides or more inlength, preferably 20 nucleotides or more, more preferably 25nucleotides, and most preferably 30 nucleotides or more. Such probes andprimers hybridize specifically to a native C. elegans fat-1polynucleotide under high stringency hybridization conditions andhybridize specifically to a native fat-1 sequence of another speciesunder at least moderately stringent conditions. Preferably, probes andprimers according to the present invention have complete sequenceidentity with the native C. elegans fat-1 sequence.

[0033] Methods for preparing and using probes and primers are described,for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”);Current Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing and Wiley-Interscience, New York, 1992 (with periodicupdates) (hereinafter, “Ausubel et al., 1992); and Innis et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, 1990. PCR-primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer™ (Whitehead Institute for Biomedical Research, Cambridge, Mass.).

[0034] Primers and probes based on the native fat-1 sequence disclosedherein can be used to confirm (and, if necessary, to correct) thedisclosed fat-1 nucleotide sequence by conventional methods, e.g., byre-cloning and sequencing a fat-1 cDNA or genomic sequence.

[0035] Nucleotide Sequence Identity. Nucleotide sequence “identity” or“similarity” is a measure of the degree to which two polynucleotidesequences have identical nucleotide bases at corresponding positions intheir sequence when optimally aligned (with appropriate nucleotideinsertions or deletions). Preferably, a fat-1 nucleotide sequence asdefined herein has at least about 75% nucleotide sequence identity,preferably at least about 80% identity, more preferably at least about85% identity, and most preferably at least about 90% identity with theC. elegans fat-1 cDNA sequence (SEQ ID NO: 1). Such a degree ofnucleotide sequence identity is considered “substantial” nucleotidesequence identity. Sequence identity can be determined by comparing thenucleotide sequences of two polynucleotides using sequence analysissoftware such as the Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, Madison,Wis.

[0036] Alternatively, two polynucleotides are substantially similar ifthey hybridize under stringent conditions, as defined below.

[0037] “Operably Linked”. A first nucleic-acid sequence is “operably”linked with a second nucleic-acid sequence when the first nucleic-acidsequence is placed in a functional relationship with the secondnucleic-acid sequence. For instance, a promoter is operably linked to acoding sequence if the promoter affects the transcription or expressionof the coding sequence. Generally, operably linked DNA sequences arecontiguous and, where necessary to join two protein coding regions, inreading frame.

[0038] “Recombinant”. A “recombinant” polynucleotide is made by anartificial combination of two otherwise separated segments of sequence,e.g., by chemical synthesis or by the manipulation of isolated segmentsof polynucleotides by genetic engineering techniques.

[0039] Techniques for nucleic-acid manipulation are well-known (see,e.g., Sambrook et al., 1989, and Ausubel et al., 1992). Methods forchemical synthesis of polynucleotides are discussed, for example, inBeaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucciet al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis ofpolynucleotides can be performed, for example, using commercialautomated oligonucleotide synthesizers.

[0040] Preparation of Recombinant or Chemically SynthesizedPolynucleotides; Vectors, Transformation, Host cells. Natural orsynthetic polynucleotides according to the present invention can beincorporated into recombinant nucleic-acid constructs, typically DNAconstructs, capable of being introduced into, replicating in, andexpressing a FAT-1 polypeptide in a host cell.

[0041] For the practice of the present invention, conventionalcompositions and methods for preparing and using vectors and host cellsare employed, as discussed, inter alia, in Sambrook et al., 1989, orAusubel et al., 1992.

[0042] A cell, tissue, organ, or organism into which has been introduceda foreign polynucleotide, such as a recombinant vector, is considered“transformed”, “transfected”, or “transgenic.” A “transgenic” or“transformed” cell or organism also includes progeny of the cell ororganism and progeny that carries the transgene, including, for example,progeny of sexual crosses between a transgenic parent and anon-transgenic parent that exhibit an altered phenotype resulting fromthe presence of a fat-1 polynucleotide construct.

[0043] Such a construct preferably is a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell.Expression vectors may include, for example; any well-known origin ofreplication or autonomously replicating sequence (ARS), expressioncontrol sequence, promoter, enhancer, secretion signal, ribosome-bindingsite, RNA splice site, polyadenylation site, transcriptional terminatorsequence, mRNA stabilizing sequence, etc., that is operable in a givenhost. Such DNA constructs are prepared and introduced into a hostcell(s) by conventional methods.

[0044] Expression and cloning vectors preferably also include aselectable or screenable marker appropriate for a given host cell ororganism. Typical selection genes encode proteins that, for example (a)confer resistance to antibiotics or other toxic substances, e.g.ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media.

[0045] The vectors containing the polynucleotides of interest can beintroduced into a host cell by any well-known method, includingelectroporation; transfection employing calcium chloride, rubidiumchloride calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as T-DNA of Agrobacterium. for plant celltransformation or a retroviral genome for animal cell transformation);etc.

[0046] The fat-1 gene is derived from C. elegans and, when expressed inplants and yeast, the gene product is biologically active. Therefore, itis expected that fat-1 can be successfully expressed and that theexpressed FAT-1 polypeptide will be active in a wide variety ofprokaryotic and eukaryotic hosts, including, but not limited to:bacteria, including Gram negative bacteria such as Escherichia coli andGram-positive bacteria such as Bacillus (e.g., B. subtilis),cyanobacteria, phytoplankton, algae, fungi (including, but not limitedto, yeast such as Saccharomyces cerevisiae and filamentous fungi),plants (including monocots and dicots) , and animals (e.g., insect,avian, and mammalian species and marine organisms such as Schizochytriumspp.).

[0047] If a host cell does not naturally produce a substrate for FAT-1,one or more substrate molecules can be provided exogenously to cellstransformed with an expressible fat-1 polynucleotide, or fat-1 can beco-expressed in cells together with one or more cloned genes that encodepolypeptides that can produce substrate compounds from compoundsavailable in such cells.

[0048] A recombinant fat-1 polynucleotide expression vector in a cellcan be used to produce a recombinant FAT-1 polypeptide that isfunctional in the cell to desaturate an ω-6 fatty acid, which isnaturally produced by the cell or that is provided exogenously to thecell, to a corresponding ω-3 fatty acid. In this way, a cell can beproduced that has a higher proportion of ω-3 fatty acids than anotherwise similar cell lacking the recombinant FAT-1 polypeptide.Alternatively, for example, an extracted lipid that includes an ω-6fatty acid can be treated with a FAT-1 polypeptide to desaturate an ω-6fatty acid to a corresponding ω-3 fatty acid. Preferably at least 10% ofan ω-6 fatty acid is desaturated to the corresponding ω-3 fatty acid,more preferably at least 20%, and most preferably at least 50%.

[0049] Nucleic-Acid Hybridization; “Stringent Conditions”; “Specific”.The nucleic-acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence, e.g., to a nativefat-1 polynucleotide.

[0050] The term “stringent conditions” is functionally defined withregard to the hybridization of a nucleic-acid probe to a targetpolynucleotide (i.e., to a particular nucleic-acid sequence of interest)by the specific hybridization procedure discussed in Sambrook et al.,1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52,9.56-9.58; Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur andDavidson, J. Mol. Biol. 31:349-370, 1968.

[0051] Regarding the amplification of a target nucleic-acid sequence(e.g., by PCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridizesubstantially only to the target nucleic-acid sequence to which a primerhaving the corresponding wild-type sequence (or its complement) wouldbind so as to produce a unique amplification product.

[0052] For hybridization of a probe or primer to a polynucleotide ofanother plant species in order to identify fat-1 homologs, preferredhybridization and washing conditions are as discussed in Sambrook etal., 1989 at 9.47-9.57, wherein “high stringency hybridizationconditions” include hybridization at 65° C. in a hybridization solutionthat includes 6×SSC and washing for 1 hour at 65° C. in a wash solutionthat includes 0.5×SSC, 0.5% SDS. “Moderate stringency” conditions aresimilar except that the temperature for the hybridization and washingsteps are performed at a lower temperature at which the probe isspecific for a target sequence, preferably at least 42° C., morepreferably at least 50° C., more preferably at 55° C., and mostpreferably at least 60° C.

[0053] The term “specific for (a target sequence)” indicates that aprobe or primer hybridizes under given hybridization conditionssubstantially only to the target sequence in a sample comprising thetarget sequence. It is expected that hybridization of a C. elegans fat-1probe or primer to genomic DNA or cDNA of another species will identifymore than one hybridizing sequence in many cases, including fat-1homologs and other sequences having substantial-sequence identity withC. elegans fat-1, particularly other desaturase genes.

[0054] Nucleic-Acid Amplification. As used herein, “amplified DNA”refers to the product of nucleic-acid amplification of a targetnucleic-acid sequence. Nucleic-acid amplification can be accomplished byany of the various nucleic-acid amplification methods known in the art,including the polymerase chain reaction (PCR). A variety ofamplification methods are known in the art and are described, interalia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: AGuide to Methods and Applications, ed. Innis et al., Academic Press, SanDiego, 1990.

[0055] Nucleotide- and Amino-Acid Sequence Variants. Using the fat-1nucleotide and amino-acid sequences disclosed herein, those skilled inthe art can create polynucleotides and polypeptides that have minorsequence variations from the corresponding native sequence.

[0056] “Variant” polynucleotides are polynucleotides containing minorchanges in a native fat-1 polynucleotide sequence, i.e., changes inwhich one or more nucleotides of a native fat-1 polynucleotide isdeleted, added, and/or substituted, preferably while substantiallymaintaining a biological activity of FAT-1. Variant polynucleotides canbe produced, for example, by standard DNA mutagenesis techniques or bychemically synthesizing the variant polynucleotide molecule or a portionthereof. Such variants preferably do not change the reading frame of theprotein-coding region of the polynucleotide and preferably encode apolypeptide having no change, only a minor reduction, or an increase inFAT-1 biological activity.

[0057] Amino-acid substitutions are preferably substitutions of singleamino-acid residues. Insertions are preferably of about 1 to 10contiguous nucleotides and deletions are preferably of about 1 to 30contiguous nucleotides. Insertions and deletions are preferablyinsertions or deletions from an end of the protein-coding or non-codingsequence and are preferably made in adjacent base pairs. Substitutions,deletions, insertions or any combination thereof can be combined toarrive at a final construct.

[0058] Preferably, variant polynucleotides according to the presentinvention are “silent” or “conservative” variants. “Silent” variants arevariants of a native fat-1 sequence or a homolog thereof in which therehas been a substitution of one or more base pairs but no change in theamino-acid sequence of the polypeptide encoded by the polynucleotide.“Conservative” variants are variants of the native fat-1 polynucleotideor an allele or homolog thereof in which at least one codon in theprotein-coding region of the polynucleotide has been changed, resultingin a conservative change in one or more amino-acid residues of thepolypeptide encoded by the polynucleotide, i.e., an amino acidsubstitution. A number of conservative amino acid substitutions arelisted below. In addition, one or more codons encoding cysteine residuescan be substituted for, resulting in a loss of a cysteine residue andaffecting disulfide linkages in the FAT-1 polypeptide. TABLE 1 OriginalResidue Conservative Substitutions Ala ser Arg lys Asn gin, his Asp gluCys ser Gln asn Glu asp Gly pro His asn; gln Ile leu, val Leu ile; valLys arg; gln; glu Met leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyrTyr trp; phe Val ile; leu

[0059] Substantial changes in function are made by selectingsubstitutions that are less conservative than those listed above, e.g.,causing changes in: (a) the structure of the polypeptide backbone in thearea of the substitution; (b) the charge or hydrophobicity of thepolypeptide at the target site; or (c) the bulk of an amino acid sidechain. Substitutions generally expected to produce the greatest changesin protein properties are those in which: (a) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) acysteine or proline is substituted for (or by) any other residue; (c) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, e.g.,glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

[0060] Polypeptides

[0061] “FAT-1 Polypeptide”. The term “FAT-1 polypeptide” (or protein)refers to a polypeptide encoded by a fat-1 polynucleotide, includingalleles, homologs, and variants of a native fat-1 polynucleotide. AFAT-1 polypeptide can be produced by the expression of a recombinantfat-1 polynucleotide or can be chemically synthesized. Techniques forchemical synthesis of polypeptides are described, for example, inMerrifield, J. Amer. Chem. Soc. 85:2149-2156, 1963.

[0062] Polypeptide Sequence Identity and Similarity. FAT-1 polypeptidesencompassed by the present invention have at least about 60% amino acidsequence similarity to the C. elegans FAT-1 polypeptide (SEQ ID NO: 2),more preferably at least about 70% similarity, more preferably at leastabout 80% similarity, and most preferably at least about 95% similarity.Even more preferable are similar degrees of amino acid sequenceidentity. Such similarity (or identity) is considered to be“substantial” similarity (or identity), although more important thanshared amino-acid sequence similarity can be the common possession ofcharacteristic structural features and the retention of biologicalactivity that is characteristic of FAT-1.

[0063] Amino acid sequence “identity” (or “homology”) is a measure ofthe degree to which aligned amino acid sequences possess identical aminoacids at corresponding positions. Amino acid sequence “similarity” is ameasure of the degree to which aligned amino acid sequences possessidentical amino acids or conservative amino acid substitutions atcorresponding positions.

[0064] Amino acid sequence similarity and identity is typically analyzedusing sequence analysis software such as the Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, Madison, Wis.). Polypeptide sequence analysissoftware matches homologous sequences using measures of homologyassigned to various substitutions, deletions, substitutions, and othermodifications.

[0065] “Isolated,” “Purified,” “Homogeneous” Polypeptides. A polypeptideis “isolated” if it has been separated from the cellular components(nucleic acids, lipids, carbohydrates, and other polypeptides) thatnaturally accompany it. Such a polypeptide can also be referred to as“pure” or “homogeneous” or “substantially” pure or homogeneous. Thus, apolypeptide that is chemically synthesized or recombinant (i.e., theproduct of the expression of a recombinant polynucleotide, even ifexpressed in a homologous cell type) is considered to be isolated. Amonomeric polypeptide is isolated when at least 60% by weight of asample is composed of the polypeptide, preferably 90% or more, morepreferably 95% or more, and most preferably more than 99% Protein purityor homogeneity is indicated, for example, by polyacrylamide gelelectrophoresis of a protein sample, followed by visualization of asingle polypeptide band upon staining the polyacrylamide gel; highperformance liquid chromatography; or other conventional methods.

[0066] It is expected that purified FAT-1 polypeptide will be useful forenzymatic conversion of ω-6 fatty acids to corresponding ω-3 fatty acidsunder conditions (e.g., detergent, salt, pH, temperature) suitable forFAT-1 enzymatic activity, for example, with FAT-1 polypeptideincorporated into liposomes or free in solution.

[0067] Protein Purification. The polypeptides of the present inventioncan be purified by any of the means known in the art. Various methods ofprotein purification are described, e.g., in Guide to ProteinPurification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, SanDiego, 1990; and Scopes, Protein Purification: Principles and Practice,Springer Verlag, New York, 1982.

[0068] Variant and Modified Forms of FAT-1 Polypeptides. Encompassed bythe FAT-1 polypeptides of the present invention are variant polypeptidesin which there have been substitutions, deletions, insertions or othermodifications of a native FAT-1 polypeptide. The variants substantiallyretain structural characteristics and biological activities of acorresponding native FAT-1 polypeptide and are preferably silent orconservative substitutions of one or a small number of contiguous aminoacid residues.

[0069] A native FAT-1 polypeptide sequence can be modified byconventional methods, e.g., by acetylation, carboxylation,phosphorylation, glycosylation, ubiquitination, and labeling, whetheraccomplished by in vivo or in vitro enzymatic treatment of a FAT-1polypeptide or by the synthesis of a FAT-1 polypeptide using modifiedamino acids.

[0070] Labeling. FAT-1 polypeptides can be labeled using conventionalmethods and reagents. Typical labels include radioactive isotopes,ligands or ligand receptors, fluorophores, chemiluminescent agents, andenzymes. Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Sambrook etal., 1989 and Ausubel et al., 1992.

[0071] Polypeptide Fragments. The present invention also encompassesfragments of a FAT-1 polypeptide that lacks at least one residue of anative full-length FAT-1 polypeptide. Preferably, such a fragmentretains FAT-1 desaturase activity, possession of a characteristicfunctional domain, or an immunological determinant characteristic of anative FAT-1 polypeptide. Immunologically active fragments typicallyhave a minimum size of 7 to 17 or more amino acids. Fragments retainingsubstantial desaturase activity are preferred, and can be obtained bydeleting one or more amino acids from the N-terminus or the C-terminusof the polypeptide, for example.

[0072] Fusion Polypeptides. The present invention also provides fusionpolypeptides including, for example, heterologous fusion polypeptides inwhich a FAT-1 polypeptide sequence is joined to a well-known fusionpartner. Such fusion polypeptides can exhibit biological properties(such as substrate or ligand binding, enzymatic activity, antigenicdeterminants, etc.) derived from each of the fused sequences. Fusionpolypeptides are preferably made by the expression of recombinantpolynucleotides that include sequences for each of the fusion partnersjoined in frame.

[0073] Polypeptide Sequence Determination. The sequence of a polypeptideof the present invention is determined by any conventional method.

[0074] Antibodies

[0075] The present invention also encompasses polyclonal and/ormonoclonal antibodies capable of specifically binding to a FAT-1polypeptide and/or fragments thereof. Such antibodies are raised againsta FAT-1 polypeptide or fragment thereof and are capable ofdistinguishing a FAT-1 polypeptide from other polypeptides, i.e., theyare FAT-1-specific.

[0076] For the preparation and use of antibodies according to thepresent invention, including various immunoassay techniques andapplications, see, e.g., Goding, Monoclonal Antibodies: Principles andPractice, 2d ed, Academic Press, New York, 1986; and Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1988. FAT-1-specific antibodies are useful, forexample in: purifying a FAT-1 polypeptide from a biological sample, suchas a host cell expressing a recombinant FAT-1 polypeptide; in cloning afat-1 allele or homolog from an expression library; as antibody probesfor protein blots and immunoassays; etc.

[0077] Such antibodies can be labeled by any of a variety ofconventional methods. Suitable labels include, but are not limited to,radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentagents, chemiluminescent agents, magnetic particles, etc.

[0078] Obtaining Alleles and Homologs of fat-1

[0079] Based upon the availability of the fat-1 cDNA sequence disclosedherein, genomic clones and alleles and homologs of the disclosed fat-1sequence can be obtained by conventional methods, e.g., by screening acDNA or genomic library with a probe that specifically hybridizes to anative fat-1 polynucleotide under at least moderately stringentconditions, by PCR or another amplification method using a primer orprimers that specifically hybridize to a native fat-1 polynucleotideunder at least moderately stringent conditions, or by identification offat-1 alleles or homologs in an expression library using FAT-1-specificantibodies. The identity of fat-1 alleles or homologs can be confirmedby application of an exogenous ω-6 fatty acid substrate to cells (e.g.,bacterial, yeast, or plant cells) in which the cloned fat-1 gene isexpressed, followed by gas chromatography analysis to determine whetherthe cloned gene converts the ω-6 substrate to the corresponding ω-3fatty acid, for example.

[0080] Probes and primers based on the fat-1 sequence disclosed hereincan also be used to obtain closely related genes having substantialnucleotide sequence identity to fat-1, e.g., other desaturase genes,including other ω-3 fatty acyl desaturase genes, by conventionalmethods.

[0081] Plant Transformation and Regeneration

[0082] Nucleic-acid constructs that include a fat-1 polynucleotide areuseful for producing transgenic plants that are capable of efficientlyconverting ω-6 fatty acids, including fatty acids having a carbon chainof greater than 18 carbons (e.g., 20, 22, or 24 carbons), to thecorresponding ω-3 fatty acids, thus producing plant cells and lipidsobtained therefrom that have an altered fatty acid profile. Such plantsinclude plants that are commonly grown for oil production, including,but not limited to, rapeseed, corn, canola, safflower, soybean,sunflower, peanut, etc.

[0083] A number of vectors suitable for stable transfection of plantcells or for the establishment of transgenic plants have been describedin, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985,supp. 1987); Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 1989; and Gelvin et al., Plant MolecularBiology Manual, Kluwer Academic Publishers, 1990.

[0084] Examples of constitutive plant promoters useful for expressingfat-1 polynucleotides include but are not limited to: the cauliflowermosaic virus (CaMV) 35S promoter, which confers constitutive, high-levelexpression in most plant tissues (see, e.g., Odel et al., Nature313:810, 1985), including monocots (see, e.g., Dekeyser et al., PlantCell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389, 1990);the nopaline synthase promoter (An et al., Plant Physiol. 88:547, 1988)and the octopine synthase promoter (Fromm et al., Plant Cell 1:977,1989).

[0085] A variety of plant gene promoters that are regulated in responseto environmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of a fat-1 polynucleotide in plant cells.Seed-specific promoters are preferred, but such regulated promoters mayalso include promoters regulated by: (1) heat (Callis et al., PlantPhysiol. 88:965, 1988); (2) light (e.g., pea rbcS-3A promoter,Kuhlemeier et al., Plant Cell 1:471, 1989; maize rbcS promoter,Schaffner and Sheen, Plant Cell 3:997, 1991; or chlorophyll a/b-bindingprotein promoter, Simpson et al., EMBO J. 4:2723, 1985); (3) hormones,such as abscisic acid (Marcotte et al., Plant Cell 1:969, 1989); (4)wounding (e.g., wunI, Siebertz et al., Plant Cell 1:961, 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or safeners.

[0086] In addition, vectors for plant expression can include additionalregulatory sequences from the 3′-untranslated region of plant genes(Thornburg et al., Proc. Natl- Acad. Sci. USA 84:744 (1987); An et al.,Plant Cell 1:115 (1989), e.g., a 3′ terminator region to increase mRNAstability of the mRNA, such as the PI-II terminator region of potato orthe octopine or nopaline synthase 3′ terminator regions.

[0087] Useful dominant selectable marker genes include genes encodingantibiotic resistance genes (e.g., resistance to hygromycin, kanamycin,bleomycin, G418, streptomycin or spectinomycin); and herbicideresistance genes (e.g., phosphinothricin acetyltransferase).

[0088] Any well-known method can be employed for plant celltransformation, culture, and regeneration in the practice of the presentinvention with regard to a particular plant species. Conventionalmethods for introduction of foreign DNA into plant cells include, butare not limited to: (1) Agrobacterium-mediated transformation(Lichtenstein and Fuller, in: Genetic Engineering, Vol 6, Rigby, ed.,London, Academic Press, 1987; and Lichtenstein and Draper, in: DNACloning, Vol II, Glover, ed., Oxford, IRI Press, 1985); (2) particledelivery (see, e.g., Gordon-Kamm et al., Plant Cell 2:603, 1990; orBioRad Technical Bulletin 1687); (3) microinjection (see, e.g., Green etal., Plant Tissue and Cell Culture, Academic Press, New York, 1987); (4)polyethylene glycol (PEG) procedures (see, e.g., Draper et al., PlantCell Physiol. 23:451, 1982); Zhang and Wu, Theor. Appl. Genet. 76:835,1988); (5) liposome-mediated DNA uptake (see, e.g., Freeman et al.,Plant Cell Physiol. 25:1353, 1984); (6) electroporation (see, e.g.,Fromm et al., Nature 319:791, 1986); and (7) vortexing methods (see,e.g., Kindle, Proc. Natl. Acad. Sci. USA 87:1228, 1990).

[0089] The term “plant” encompasses any higher plant and progenythereof, including monocots (e.g., lily, corn, rice, wheat, barley,etc.), dicots (e.g., tomato, potato, soybean, cotton, tobacco, etc.),and includes parts of plants, including reproductive units of a plant(e.g., seeds, fruit, flowers, etc.)

[0090] A “reproductive unit” of a plant is any totipotent part or tissueof the plant from which one can obtain a progeny of the plant,including, for example, seeds, cuttings, tubers, buds, bulbs, somaticembryos, cultured cells (e.g., callus or suspension cultures), etc.

[0091] Transformation of Algal Cells

[0092] Nucleic-acid constructs that include a fat-1 polynucleotide arealso useful for recombinant expression in algal cells, includingplankton, that are capable of efficiently converting ω-6 fatty acids tothe corresponding ω-3 fatty acids. Manipulation of algal cells ingeneral is discussed, for example, in Dunahay et al., Appl. Biochem.Biotechnol. 57/58:223-231, 1996 and Dunahay et al., J. Phycol.31:1004-1012, 1995.

[0093] The invention will be better understood by reference to thefollowing Examples, which are intended to merely illustrate the bestmode now known for practicing the invention. The scope of the inventionis not to be considered limited thereto.

EXAMPLES Example 1

[0094] The Cloning and Sequencing of a cDNA Encoding fat-1, an AnimalOmega-3 Desaturase

[0095] In Arabidopsis there are seven membrane desaturases. Thebiochemistry and function of these membrane desaturases has beenfacilitated by the availability of mutants with altered fatty acidcompositions (Browse et al., Science 227:763-765, 1985) (Browse andSomerville, In: Arabidopsis, Cold Spring Harbor Press, New York, 1994,pp. 881-912). Genes encoding desaturases in Arabidopsis have been cloned(Arondel et al., Science 258:1353-1355, 1992; Yadav et al., PlantPhysiol. 103:467-476, 1993; Yadav et al. in: Biochemistry and MolecularBiology of Membrane and Storage Lipids of Plants, ed. Murala andSomerville, American Society of Plant Physiologists, 1993, pp. 60-66;Okuley et al., Plant Cell 6:147-158, 1994). All the plant membrane-bounddesaturases use complex glycerolipids rather than acyl-CoAs assubstrates. This finding, together with data indicating that the Δ5desaturase from rat liver acts on glycerolipids (Pugh and Kates, J.Biol. Chem. 252:68-73, 1977), suggested the possibility that themajority of animal desaturases also catalyze glycerolipid-linkeddesaturation. However, there has been little progress in characterizingother animal desaturases. In animals, fatty acid desaturases catalyzekey reactions in the synthesis of arachidonic acid and otherpolyunsaturated fatty acids.

[0096]C. elegans elaborates a wide range of polyunsaturated fatty acids,including arachidonic (20:4, ω-6) and eicosapentaenoic acids (20:5, ω3)from very simple precursors available in the diet of the organism(Hutzell and Krusberg, Comp. Biochem. Physiol. 73B:517-520, 1982;Satouchi et al., Lipids 28:837-840, 1993). The ω-3 fatty acids(Δ9,12,15-18:3; Δ8,11,14, 17-20:4 and Δ5,8,11,14,17-20:5) account for17% of the total fatty acids in C. elegans (Hutzell and Krusberg, Comp.Biochem. Physiol. 73B:517-520, 1982) and 20:5 ω-3 is the major fattyacid in phosphatidylcholine from this organism (Satouchi et al., Lipids28:837-840, 1993). These lipids are produced even when the worms aregrown exclusively on E. coli, which provides only saturated andmonounsaturated fatty acids (Satouchi et al., Lipids 28:837-840, 1993).Evidently, C. elegans must contain all the enzymes required for thesynthesis of these highly unsaturated acyl groups.

[0097] We searched the National Center for Biotechnology Information's(NCBI) peptide sequence data base using a BLAST server with the peptidesequences of the Arabidopsis thaliana FAD2, FAD6 and FAD7 fatty aciddesaturases as queries. (The GenBank accession numbers for thecorresponding Arabidopsis cDNAs are L26296, U09503 and L22931,respectively.) The highest scoring C. elegans expressed sequence tag(EST) clones were NCBI-5443, NCBI-5881 and NCBI-5049. (The correspondingΔ4 10 GenBank accession numbers were Z14935, M88884 and Z14543,respectively.) An alignment of cDNA sequences revealed a common identityof 301 bp among all three clones, indicating that the three ESTsoriginated from a single gene. The greatest amount of sequence data (486bp) was available from EST clone NCBI-5881. This clone was requestedfrom its origin at the C. elegans genome Sequencing Center, WashingtonUniversity School of Medicine, St. Louis, Mo., with its original sourceidentifier (CEL10ell). Upon receipt of CEL10ell, its identity wasconfirmed by partial sequencing.

[0098] In order to obtain a full-length cDNA corresponding to CEL10ell,the cDNA insert was released by double digestion with HindIII and SacI,gel purified, and labeled with ³²P-dCTP using a random priming kit(Promega, Madison, Wis.). The denatured probe was used to screen a mixedstage C. elegans cDNA lambda phage (Uni-Zap XR) library (Stratagene, LaJolla, Calif.). Nucleic acid hybridizations and high stringency washeswere performed as described (Amasino, Anal. Biochem. 152:304-307, 1986).Thirteen hybridizing plaques were visualized by autoradiography. Thelongest eight clones were all approximately 1.4 kb in length as judgedby agarose gel electrophoresis. Positive clones were isolated andexcised from the phage vector according to the manufacturer's protocolto yield pBluescript™ plasmids.

[0099] The plasmid clone with the longest insert, pCE8, was fullysequenced in both directions and found to contain a 1,410 bp cDNAinsert. Sequence analysis was carried out using the programs availablein the GCG package (Devereaux et al., Nucl. Acids Res. 12:387-395, 1984)using default settings for parameters unless otherwise indicated. Thissequence was deposited in GenBank under Accession Number L41807.

[0100] The nucleotide sequence of the fat-1 cDNA (1391 nt) and thededuced FAT-1 amino-acid sequence are shown in FIG. 1 (and SEQ ID NO: 1and 2).

[0101] The DNA sequence corresponding to the open reading frame of thefat-1 cDNA was used to search the database of the C. elegans genomesequencing project using the BLAST server (Waterston and Sulston, Proc.Natl. Acad. Sci. USA 92:10836-10840, 1995). No homologous sequence wasfound (highest BLAST score: 145; p=0.034), indicating that the fat-1genomic sequence had not yet been included in this project.

[0102] The cDNA insert in pCE8 contained an open reading frame thatwould be expected to encode a protein of 402 amino acids, with amolecular mass of 46.4 kD. Sequence comparisons were made using theprograms of the Genetics Computer Group Package (Devereaux et al., Nucl.Acids Res. 12:387-395, 1984). The predicted amino acid sequence of theprotein showed several regions of common homology with the predictedsequences of the FAD2 and FAD3 desaturases of Arabidopsis. Alignmentwith FAD2 revealed 35% sequence identity and 61% similarity (i.e.,including both identical amino acid residues and conservativesubstitutions). Alignment with FAD3 indicated 32% sequence identity and54% similarity. The FAD2 and FAD3 genes are known to encode enzymes thatdesaturate oleate (FAD2) or linoleate (FAD3) esterified tophosphatidylcholine of the endoplasmic reticulum (Arondel et al.,Science 258:1353-1355, 1992; Okuley et al., Plant Cell 6:147-158, 1994).Within a tripartite alignment of the three sequences are 69 residuescommon to all three sequences, including eight histidines (amino acids123, 127, 159, 162 163, 324, 327 and 328 in the C. elegans sequence)whose presence and locations are highly conserved among all the membranedesaturases (Okuley et al., Plant Cell 6:147-158, 1994). These findingsstrongly indicate that the gene represented by the pCE8 cDNA encodes afatty acid desaturase or a related enzyme function. We have designatedthis gene fat-1 (Fatty Acid Metabolism-1).

[0103] It is somewhat surprising that the C. elegans gene shows asimilar amino acid sequence homology to each of the two Arabidopsisdesaturases, especially in view of the fact that the FAD2 and FAD3sequences are relatively divergent, with only 37% common amino acididentity (okuley et al., Plant Cell 6:147-158, 1994). From thesecomparisons alone, it is difficult to deduce whether the fat-1 gene islikely to represent a Δ12 desaturase (like FAD2), an ω-3 desaturase(like FAD3), or a more distantly related enzyme.

[0104] In contrast with the identity found between the deduced FAT 1polypeptide sequence and FAD2 and FAD3 of Arabidopsis, there was only17-23% homology to the yeast and rat genes that encode 18:0-CoAdesaturases, only slightly above the level from comparisons withentirely unrelated genes. For this reason, it is unlikely that adatabase search using an 18:0-CoA desaturase as the query could haveidentified the fat-1 sequence.

[0105] The predicated protein sequence of the fat-1 gene productincludes the three histidine-rich sequences that are highly conservedamong all the membrane-bound fatty acid desaturases and that arebelieved to be the residues that coordinate the diiron-oxo structure atthe active site of these enzymes (Shanklin et al., Biochemistry33:12787-12794, 1994; Stukey et al., J. Biol. Chem. 265:20144-20149,1990). Furthermore, two long stretches (>40 residues each) ofhydrophobic residues are present (80 to 124 and 229 to 284). The lengthof these stretches and their positions relative to the conservedhistidine sequences are similar to other desaturases. Therefore, theFAT-1 protein could conform with the model proposed by Stukey et al.(Stukey et al., J. Biol. Chem. 265:20144-20149, 1990), in which the bulkof the protein is exposed on the cytosolic face of the endoplasmicreticulum, while two membrane-traversing loops (each comprised of twomembrane-spanning, α-helical segments) lock the protein into thebilayer. In common with many, though not all, of the proposedendoplasmic reticulum desaturases, the FAT-1 protein contains acarboxy-terminal motif (KAKAK) that conforms to a consensus retentionsignal for transmembrane proteins in the endoplasmic reticulum (Jacksonet al., EMBO J. 9:3153-3162, 1990).

[0106] These features are consistent with FAT-1 being a member of themembrane-bound desaturase/hydroxylase family of diiron-oxo proteins(Shanklin et al., Biochemistry 33:12787-12794, 1994). However, the FAT-1sequence shows equal homology to both the Δ12 glycerolipid desaturaseencoded by FAD2 and the ω-3 glycerolipid desaturase encoded by FAD3.

Example 2

[0107] Expression of fat-1 in Arabidopsis and Characterization of ItsFunction

[0108] To determine which class of reaction is catalyzed by FAT-1 and toexplore the substrate chainlength and regiochemical specificities of theenzyme it was necessary to use heterologous expression of a fat-1 cDNAin a host that contained potential fatty acid substrates. BothEscherichia coli and Saccharomyces cerevisiae, which are two commonlaboratory hosts for heterologous expression, possess a very limitedrange of endogenous desaturation activities and hence fatty acidcompositions. By contrast, plants possess both a wider range ofdesaturase activities and fatty acids that are potential substrates fora desaturase of unknown function. In addition, the lipid and fatty acidmetabolism of plants, especially Arabidopsis, have been wellcharacterized. These features make Arabidopsis a more attractive hostfor transgenic studies of putative eukaryotic fatty acid desaturasesthan either E. coli or S. cerevisiae.

[0109] In higher plants, desaturases have been characterized from twocellular compartments. Enzymes localized to the chloroplast (or plastid)use soluble ferredoxin as the electron donor for the reaction (Schmidtet al., Plant Mol. Biol. 26:631-642, 1994; Heinz, in Lipid Metabolism inPlants, ed., CRC Press, Boca Raton, Fla., 1993, pp. 33-89). Enzymeslocalized to the endoplasmic reticulum (including the FAD2 and FAD3 geneproducts) are similar to known yeast and animal desaturases inasmuch asthey rely on cytochrome b₅ and cytochrome b₅ reductase to supplyelectrons from NAD (P) H (Heinz, in Lipid Metabolism in Plants, ed., CRCPress, Boca Raton, Fla., 1993, pp. 33-89). Mutants deficient in each ofthe major desaturases are available in Arabidopsis (Browse andSomerville, in Arabidopsis, ed. Cold Spring Harbor Press, New York, pp.881-912, 1994). Genes that encode the 18:0-CoA desaturases from yeastand mammals have been expressed in plants and shown to alter the fattyacid compositions of the plant tissues (Polashok et al., Plant Physiol.100:894-901, 1992; Grayburn et al., Bio/Technology 10:675-677, 1992).These considerations indicated that Arabidopsis is a suitableheterologous system to study the expression and function of the fat-1gene.

[0110] In order to produce a fat-1 gene construct for plant expression,the cauliflower mosaic virus (CaMV) 35S promoter/nopaline synthaseterminator cassette of Baulcombe et al. (Baulcombe et al., Nature321:446-449, 1986) was cloned into the XbaI/EcoRI sites of pBIN400(Spychalla and Bevan, In: Plant Tissue Culture Manual: Fundamentals andApplications, ed. Lindsay, Kluwer Academic Publishers, Dordrecht, Vol.B11, 1993, pp. 1-18) to make the binary transformation vector pBIN420.The cDNA insert of pCE8 was released with a EcoRI/KpnI double digest,end-filled with Klenow fragment, and blunt-ligated into the SmaI site ofpBIN420 to make pBIN420-CE8. These vectors contain the NPTII gene withintheir T-DNA, thus conferring kanamycin resistance to transgenic plants.

[0111] The Columbia ecotype of the wild-type line of Arabidopsisthaliana (L.) Heynh. was used for plant transformation. The binaryvector pBIN420-CE8 was introduced into the Agrobacterium strain PC27G0by the freeze-thaw method (Holsters et al., Mol. Gen. Genet.163:181-187, 1978). Agrobacterium-mediated transformation wasaccomplished with the in planta vacuum-infiltration method (Bouchez etal., C. R. Acad. Sci. Paris 316:1188-1193, 1993). Primary generationtransformed seeds were selected on plates containing Murashige and Skoogbasal salts (4.3 g/L), 1% (w/v) sucrose, 0.8% (w/v) Bacto-Agar, 200 mg/Lcarbenicillin, and 50 mg/L kanamycin, and was adjusted to pH 5.8 withKOH. In vitro roots were grown from sterilized seeds placed on verticalplates at 23° C. under continuous illumination (50-100 micromol quantam⁻²s⁻¹). The media for in vitro roots contained Gamborg B5 salts (3.1g/L), 2% (w/v) glucose, and 0.2% Phytagel™ (Sigma, St. Louis, Mo.), andwas adjusted to pH 5.8 with KOH.

[0112] Five individual transformants were obtained and allowed to setseed. Lines #9.7 and #10.5 were selected for further analysis bySouthern and Northern blotting using the HindIII/SacI fragment of pCE8as a probe on the RNA and DNA blots. Probe labeling, hybridizations andwashings were as described above for cDNA library screens.

[0113] For Southern blots, genomic DNA was isolated from lines #9.7 and#10.5 according to the method of Dellaporta et al. (Dellaporta et al.,Plant Mol. Biol. Rep. 1:19-21, 1983), restricted with BamHI, separatedby agarose gel-electrophoresis, and alkaline blotted to nylon membranes.The Southern blot confirmed the presence of at least one copy of thetransgene in line #9.7 and at least two copies in line #10.5.

[0114] For Northern blots, total RNA was extracted from leaves accordingto the method of Verwoerd et al. (Verwoerd et al., Nucl. Acids Res.17:2362, 1989). Twenty-five micrograms of total RNA was separated on1.2% agarose-formaldehyde gels and blot transferred to nylon membranes.Northern blots of total RNA from plants of the two lines and wild-typeArabidopsis showed that the appropriate fat-1 transcript accumulated inboth transgenic lines. Plants from line #9.7 consistently producedhigher transcript levels than line #10.5.

[0115] Characterization of Arabidopsis lipid mutants has indicated thatlesions in the fad2 and fad3 genes are partly masked in leaf tissue byaction of the chloroplast desaturases (encoded by FAD6, FAD7 and FAD8).For this reason, a first attempt to determine the function of the fat-1gene product was made by analyzing the overall fatty-acid composition ofroot tissues from wild-type and fat-1 transgenic plants. The data inTable 2 show very large increases in the proportion of 18:3 in bothtransgenic lines compared with wild-type Arabidopsis. These increaseswere accompanied by concomitant decreases in the proportion of 18:2 butno significant changes in the levels of any other fatty acid. Thealterations in root fatty-acid composition induced by expression of theC. elegans fat-1 gene are comparable to those observed by overexpressionof the plant FAD3 gene (Arondel et al., Science 258:1353-1355, 1992).The FAT1 protein is thus operating as an efficient ω-3 desaturase inArabidopsls. TABLE 2 Composition (mol %) of total fatty acids from invitro grown roots of the wild type (WT) and two transgenic lines (#9.7and #10.5) of Arabidopsis expressing a C. elegans fat-1 cDNA¹ Genotype16:0 16:1(c) 18:0 18:1 18:2 18:3 WT 16.8a 1.2a 1.4a 22.7a 39.3a 18.0b #9.7 17.8a 1.2a 2.2a 21.7a 22.7b 33.7a #10.5 17.5a 1.0a 1.6a 20.9a23.7b 34.5a

[0116] In untreated Arabidopsis, linolenic acid (Δ9,12,15-18:3) is theonly significant product resulting from fat-7 expression. However, C.elegans contains a wider range of PUFAs than does Arabidopsis. Three ω-3fatty acids are present in the membrane lipids of the worm, of whichlinolenic acid is the least abundant (0.15% of total fatty acids). The20-carbon fatty acids Δ8,11,14,17-eicosatetraenoic acid (an isomer ofarachidonic acid) and Δ5,8,11,14,17-eicosapentaenoic acid (the expectedproduct of ω-3 desaturation of arachidonic acid) account for 7.7% and8.7% of total fatty acids, respectively (Hutzell and Krusberg, Comp.Biochem. Physiol. 73B:517-520, 1982).

[0117] Exogenous fatty acids applied to Arabidopsis leaves as sodiumsoaps are readily taken up and incorporated into membrane glycerolipidsto levels that correspond to 2-5% of the total leaf lipids (McConn andBrowse, Plant Cell 8:403-416, 1996). To test whether the fat-1-encodeddesaturase is likely to be involved in synthesis of the 20-carbon ω-3fatty acids in C. elegans, wild-type and transgenic Arabidopsis plantswere sprayed once a day for twenty days with solutions of the sodiumsalts of arachidonic acid (Δ5,8,11,14-20:4) or a honogamma linolenicacid (Δ8,11,14-20:3).

[0118] For exogenous fatty-acid treatments, plants were grown in agrowth chamber at 20° C. on a 12 h day/night cycle. Fatty-acidtreatments began when the plant rosettes reached approximately 2 cm indiameter. Sodium soaps of homogamma-linolenic acid (Δ8,11,14-20:3) andarachidonic acid (Δ5,8,11,14-20:4) (NuCheck Prep, Elysian, Minn.) weremade to a 0.1% aqueous solution and frozen in 5 mL aliquots. Plants weresprayed daily at the beginning of the dark period using a perfumeatomizer. Groups of fifteen plants were sprayed with 5 mL of soapsolution for 20 consecutive days.

[0119] Methods for extraction and separation of lipids, and for thepreparation of fatty acid methyl esters have been described previously(Miquel and Browse, J. Biol. Chem. 267:1502-1509, 1992). Analysis offatty acid methyl esters by gas chromatography was carried out using a15 m×0.53 mm Supelcowax column (Supelco, Bellefonte, Pa.) with flameionization detection. The initial column temperature of 160° C. was heldfor 1 min, then raised at 20° C./min to 190° C., followed by a ramp of5° C./min to 230° C. The final temperature was held for 5 min. Whenwild-type and fat-1 transgenic plants were sprayed with exogenous fattyacids, the peaks for the ω-6 substrates, Δ8,11,14-20:3, Δ5,8,11,14-20:4an ω-3 desaturation products Δ8,11,14,17-20:4 and Δ5,8,11,14,17-20:5were identified based on their coelution with authentic standards(NuCheck Prep, Elysian, Minn.) and on the results of gaschromatography-mass spectrometry (GC-MS) analysis. For this analysis,fatty acid methyl esters derived from phosphatidylcholine were separatedon a 30 m×0.2 mm AT1000 column (Alltech Assoc., Deerfield, Ill.) in aHP6890 Instrument (Hewlett-Packard, Avondale, Pa.). Oven temperature atinjection was 50° C. and this was increased at 5° C./min to 230° C.,then held at 230° C. for 10 min. Criterion for identification ofΔ5,8,11,14,17-20:5 in phosphatidylcholine from fat1 transgenic plantswere: (1) the identification of a mass peak at m/z=316, whichcorresponds to the expected molecular ion, and (2) a retention time(36.11 min) and fragmentation pattern identical to those of theauthentic Δ5,8,11,14,17-20:5 standard. No commercial standard wasavailable for Δ8,11,14,17-20:4. A fatty.acid methyl ester present infat-1 transgenic plants sprayed with Δ8,11,14-20:3, but not in wild-typecontrol plants, had a retention time of 35.74 min during GC-MS. Thiscompound showed a mass peak at m/z=318 (the expected molecular ion for20:4) and a fragmentation pattern very similar to that of the authenticΔ5,8,11,14-20:4 standard. The retention time of Δ5,8,11,14-20:4 was35.04 min for both the authentic standard and for the methyl estersrecovered from plants sprayed with soaps of this isomer. Therefore, itwas concluded that the new compound detected only in fat-1 transgenicplants sprayed with Δ8,11,14-20:3 was an isomer of 20:4 and mostprobably Δ8,11,14,17-20:4.

[0120] Analyses of total leaf lipids indicated that the exogenouslysupplied fatty acids were incorporated at levels of 1-3% of the totalfatty acids. There was extensive incorporation into phosphatidylcholine,which is the major lipid of the endoplasmic reticulum and the majorsubstrate for the plant 18:1 and 18:2 desaturases (Miquel and Browse, J.Biol. Chem. 267:1502-1509, 1992; Browse et al., J. Biol. Chem.268:16345-16351, 1993). In wild-type leaves, the peak corresponding toΔ5,8,11,14-20:4 or Δ8,11,14-20:3 accounted for approximately 3-5% of thetotal fatty acids in phosphatidylcholine, but there was no detectableconversion of either of these fatty acids to their ω-3 unsaturatedderivatives. By contrast, in leaves from plants expressing the fat-1cDNA, the peaks corresponding to the exogenously-supplied ω-6 fattyacids were substantially replaced by peaks that correspond to theexpected ω-3 desaturated products, Δ5,8,11,14,17-20:5 andΔ8,11,14,17-20:4. Thus, in contrast to the Arabidopsis FAD3 geneproduct, the C. elegans FAT-1 protein is a desaturase that acts on arange of ω-6 fatty acid substrates. These results demonstrate that thefat-1 gene encodes an ω-3 desaturase that is able to carry out the finalstep in the synthesis of all these fatty acids.

Example 3

[0121] Relative Efficiencies of the FAT-1 and FAD-3 Desaturases

[0122] A fat-1 cDNA confers to Arabidopsis plants the ability todesaturate 20:3, ω-6 and 20:4, ω-6 fatty acyl groups to thecorresponding ω-3 products (Δ8,11,14,17-20:4 and Δ5,8,11,14,17-20:5,respectively). The absence of detectable levels of these ω-3 fatty acidsfrom untransformed Arabidopsis tissues suggests that the endogenousplant ω-3 desaturases (the FAD3, FAD7 and FAD8 enzymes in Arabidopsis)have little or no ability to desaturate 20-carbon substrates. However,the fat-1 cDNA is highly expressed in the transgenic plant line #9.7.

[0123] To more accurately compare the relative efficiencies of the FAT-1and FAD3 desaturases, we used a transgenic Arabidopsis line(wild-type:pTiDES3) in which the FAD3 gene is overexpressed to a highdegree (Arondel et al., Science 258:1353-1355, 1992). Proportions of18:2 and 18:3 in the root fatty acid composition produced in this lineare altered to a slightly greater degree than in line #9.7, indicatingthat the FAD3-overexpressing line contains a somewhat higher activityfor ω-3 desaturation of 18:2 fatty acyl groups. However, when plants ofthe wild-type:pTiDES3 line were supplied with exogenous 20:4, ω-6 fattyacids using the protocol described above, less than 25% of this compoundwas converted to 20:5, ω-3 as judged by fatty acid analysis ofphosphatidylcholine purified from leaf tissue of the sprayed plants. Thelow extent of conversion confirms that the enzyme encoded by the fat-1cDNA is considerably more efficient than the plant FAD3 enzyme when20:4, ω-6, is the substrate for ω-3 desaturation.

[0124] The ability of Arabidopsis plants to take up exogenous fattyacids provided us with a means to extend the biochemicalcharacterization of the FAT-1 desaturase by showing that all the 18- and20-carbon ω-6 fatty acids normally present in C. elegans are recognizedas its substrates, as well as 22:5, ω-6 (i.e., Δ4,7,10,13,16-22:5). TheFAD2 and FAD3 desaturases are known to use membrane glycerolipids, notacyl-CoAs, as substrates (Miquel and Browse, J. Biol. Chem.267:1502-1509, 1992; Browse et al., J. Biol. Chem. 268:16345-16351,1993). The high efficiency with which the FAT-1 enzyme desaturates thd18:2 of Arabidopsis-membrane lipids and the high homology of FAT-1 toFAD2 and FAD3 strongly suggest that FAT-1 is also a glycerolipiddesaturase. In this respect the enzyme is similar to the Δ5 desaturaseactivity described in rat liver (Pugh and Kates, J. Biol. Chem.252:68-73, 1977). Other desaturases required for the synthesis ofarachidonic acid in mammals may also use membrane phospholipids as theirsubstrates.

Example 4

[0125] fat-1 Transgenic Arabidopsis Plants Desaturate Δ4,7,10,13,16-22:5to Δ4,7,10,13,16,19-22:6

[0126] The 22-carbon fatty acids docosapentaenoic acid (22:5, ω-6) anddocosahexaenoic acid (22:6, ω-3) also have important dietary andpharmaceutical uses. For many applications, 22:6, ω-3 is the moredesirable product. Most sources of 22-carbon highly unsaturated fattyacids contain both 22:5, ω-6 and 22:6, ω-3. We therefore investigatedwhether FAT-1 could desaturate 22:5, ω-6 to the ω-3 product.

[0127] For this purpose, plants of the transgenic Arabidopsis line #9.7,which express FAT-1, were grown together with control wild-type plantsat 24° C. with continuous illumination under fluorescent lights (150μmol quanta/m²/s). Sets of 15 leaves from 20-day-old wild-type or fat-1transgenic plants were harvested and placed in 2-inch diameter petridishes. To each petri dish was added either 4 mL of an aqueous solutioncontaining 1% (v/v) dimethylsulfoxide and 0.025% (wt/v) of the potassiumsoap of 22:5 ω-6 fatty acid or 4 mL of a similar solution lacking the22:5 ω-6 soap. Each dish was covered with a single layer of absorbenttissue to ensure good contact between the solution and the leaves,closed, covered with aluminum foil, and incubated in the dark for fourhours. After the solution was removed, the leaves were rinsed severaltimes with distilled water, then covered with 4 mL of water andincubated under fluorescent lights (150 μmol quanta/m²/s) for anadditional 24 hours before lipid extraction.

[0128] Methods for extraction and separation of lipid classes and forthe preparation of fatty acid methyl esters have been describedpreviously (Miquel and Browse, J. Biol. Chem. 267:1502-1509, 1992).Analysis of fatty acid methyl esters by gas chromatography was carriedout using a 15 m×0.5 mm Supelcowax column (Supelco, Bellefonte, Pa.)with flame ionization detection. The initial column temperature of 160°C. was held for 0.5 min, then raised at 20° C./min to 190° C. andthereafter at 5° C./min to 215° C. This final temperature was held for10 min.

[0129] The 22:5, ω-6 fatty acid was prepared from lipids of the marineorganism Schizochytrium. Schizochytrium lipids (100 mg) were dissolvedin 1 mL tetrahydrofuran and converted to fatty acid methyl esters asdescribed (Miquel and Browse, J. Biol. Chem. 267:1502-1509, 1992). The22:5 methyl ester was separated from other components by chromatographyon silica gel G plates that had been dipped in a solution of 5%AgNO₃+0.01% rhodamine B in acetonitrile using a solvent containinghexane:diethyl ether 40:60 (v/v). Fatty acid methyl ester bands werevisualized under ultraviolet light and the 22:5 (second band from thebottom of each plate) was scraped into a screw-cap tube. Water (4 mL),methanol (10 mL) and chloroform (10 mL) were added to the silica gel.The mixture was filtered through glass wool and the silica gel rinsedwith 5 mL of chloroform:methanol:water (1:1:0.1, v/v/v). The combinedfiltrate was separated into two phases by addition of 4 mL H₂O. The22:5-methyl ester was recovered with the lower phase, reduced to drynessunder a stream of nitrogen, redissolved in hexane, and stored underargon at −20° C. To prepare the sodium soap of 22:5, a sample of thehexane solution was dried in a screw-cap tube under a stream ofnitrogen, redissolved in 0.3 mL of 1 M KOH in 95% aqueous methanol,sealed under argon, and heated to 80° C. for 90 minutes. After cooling,the preparation was diluted with 2 mL of water and extracted with 2 mLof hexane to remove non-saponifiable lipids. After removal of the hexanephase, the aqueous solution was titrated to pH 10 using 0.1 M HCl. Fattyacid methyl esters from a sample of this solution were analyzed by gaschromatography to confirm the identity of the 22:5 potassium soap and tocalculate the concentration of the soap.

[0130] There was extensive incorporation of the exogenous 22:5, ω-6fatty acid into phosphatidylcholine and phosphatidylethanolamine, whichare the major phospholipids of the endoplasmic reticulum and otherextrachloroplast membranes of plant cells. Phosphatidylcholine is themajor lipid substrate for the plant 18:1 and 18:2 desaturases (Miqueland Browse, J. Biol. Chem. 278:1502-1509, 1992; Browse et al., J. Biol.Chem. 268:16345-16351, 1993). In wild-type leaves, the peakcorresponding to Δ4,7,10,13,16-22:5 accounted for approximately 2-4% ofthe total fatty acids in phosphatidylcholine, but there was nodetectable conversion of this fatty acid to the ω-3 unsaturatedΔ4,7,10,13,16,19-22:6. By contrast, in leaves of plants expressing thefat-1 cDNA, the peak corresponding to 22:5, ω-6 was substantiallyreplaced by a peak corresponding to the expected ω-3 unsaturatedproduct, Δ4,7,10,13,16,19-22:6. Data for three independent experimentsare shown in Table 3. The identity of the 22:6, ω-3 product wasconfirmed by GC-MS as described above to show that the retention time,molecular ion peak (m/z=342), and fragmentation pattern of the fattyacid isolated from fat-1 transgenic plants corresponded to those ofgenuine Δ4,7,10,13,16,19-22:6.

[0131] These results demonstrate that the FAT-1 protein, expressed in aheterologous host from a fat-1 cDNA, efficiently convertsΔ4,7,10,13,16-22:5 to Δ4,7,10,13,16,19-22:6.

[0132] We also supplied 22:5, ω-6 (0.025% w/v as the potassium soap inaqueous solution containing 1% v/v dimethylsulfoxide) to liveCaenorhabditis elegans growing in liquid culture. The C. elegansincorporated the exogenous fatty acid into phospholipids and convertedit to 22:6, ω-3. TABLE 3 Metabolism of 22:5, ω-6 fatty acid in wild-typeArabidopsis and transgenic Arabidopsis of line #9.7 expressing a fat-1cDNA. Results are the amounts of 22:5, ω-6 and 22:6, ω-3 inphosphatidylcholine expressed as a percentage of the total fatty acidsin this lipid. 22:5 22:6 Total % conversion* Experiment 1 wild-typewater control 0.00 0.00 0.00 — fat-1 water control 0.00 0.00 0.00 —wild-type +22:5 3.25 0.00 3.25 0 fat-1 +22:5 1.35 1.82 3.17 57Experiment 2 wild-type +22:5 2.67 0.00 2.67 0 fat-1 +22:5 0.57 2.31 2.8880 Experiment 3 wild-type +22:5 2.09 0.00 2.09 0 fat-1 0.36 1.67 2.03 82

Example 5

[0133] Expression of FAT-1 in Yeast

[0134] A fat-1 cDNA was incorporated into the pYX232 yeast expressionvector (Novagen Inc., 597 Science Dr., Madison, Wis. 53711) in a senseorientation at the multicloning site (to create plasmid pYX232:fat-1) sothat it could be expressed in yeast (Saccharomyces cerevisiae) cellsunder control of the triosephosphate isomerase promoter. The fat-1vector construct was transformed into yeast cells (strain YRP685), inwhich an ω-6 fatty acyl substrate, Δ9,12-18:2, was available. Gaschromatography analysis of yeast cells derived from this transformationexperiment contained approximately 1% of their total fatty acids as 18:3compared with less than 0.55 in control cells that did not containpYX232:fat-1. This increase in accumulation of 18:3 indicates that thefat-1 cDNA encodes a product that is able to act as an ω-3 fatty aciddesaturase in yeast. One can readily increase the level of ω-3desaturation in yeast cells using the same fat-1 coding sequence butemploying, for example, different combinations of well-known promotersand yeast strains.

[0135] This invention has been detailed both by example and by directdescription. It should be apparent that one having ordinary skill in therelevant art would be able to surmise equivalents to the invention asdescribed in the claims which follow but which would be within thespirit of the foregoing description. Those equivalents are to beincluded within the scope of this invention.

1 2 1391 base pairs nucleic acid double stranded linear 1 CAAGTTTGAG GT12 ATG GTC GCT CAT TCC TCA GAA GGG TTA TCC GCC ACG GCT CCG GTC 57 MetVal Ala His Ser Ser Glu Gly Leu Ser Ala Thr Ala Pro Val 5 10 15 ACC GGCGGA GAT GTT CTG GTT GAT GCT CGT GCA TCT CTT GAA GAA 102 Thr Gly Gly AspVal Leu Val Asp Ala Arg Ala Ser Leu Glu Glu 20 25 30 AAG GAG GCT CCA CGTGAT GTG AAT GCA AAC ACT AAA CAG GCC ACC 147 Lys Glu Ala Pro Arg Asp ValAsn Ala Asn Thr Lys Gln Ala Thr 35 40 45 ACT GAA GAG CCA CGC ATC CAA TTACCA ACT GTG GAT GCT TTC CGT 192 Thr Glu Glu Pro Arg Ile Gln Leu Pro ThrVal Asp Ala Phe Arg 50 55 60 CGT GCA ATT CCA GCA CAC TGT TTC GAA AGA GATCTC GTT AAA TCA 237 Arg Ala Ile Pro Ala His Cys Phe Glu Arg Asp Leu ValLys Ser 65 70 75 ATC AGA TAT TTG GTG CAA GAC TTT GCG GCA CTC ACA ATT CTCTAC 282 Ile Arg Tyr Leu Val Gln Asp Phe Ala Ala Leu Thr Ile Leu Tyr 8085 90 TTT GCT CTT CCA GCT TTT GAG TAC TTT GGA TTG TTT GGT TAC TTG 327Phe Ala Leu Pro Ala Phe Glu Tyr Phe Gly Leu Phe Gly Tyr Leu 95 100 105GTT TGG AAC ATT TTT ATG GGA GTT TTT GGA TTC GCG TTG TTC GTC 372 Val TrpAsn Ile Phe Met Gly Val Phe Gly Phe Ala Leu Phe Val 110 115 120 GTT GGACAC GAT TGT CTT CAT GGA TCA TTC TCT GAT AAT CAG AAT 417 Val Gly His AspCys Leu His Gly Ser Phe Ser Asp Asn Gln Asn 125 130 135 CTC AAT GAT TTCATT GGA CAT ATC GCC TTC TCA CCA CTC TTC TCT 462 Leu Asn Asp Phe Ile GlyHis Ile Ala Phe Ser Pro Leu Phe Ser 140 145 150 CCA TAC TTC CCA TGG CAGAAA AGT CAC AAG CTT CAC CAT GCT TTC 507 Pro Tyr Phe Pro Trp Gln Lys SerHis Lys Leu His His Ala Phe 155 160 165 ACC AAC CAC ATT GAC AAA GAT CATGGA CAC GTG TGG ATT CAG GAT 552 Thr Asn His Ile Asp Lys Asp His Gly HisVal Trp Ile Gln Asp 170 175 180 AAG GAT TGG GAA GCA ATG CCA TCA TGG AAAAGA TGG TTC AAT CCA 597 Lys Asp Trp Glu Ala Met Pro Ser Trp Lys Arg TrpPhe Asn Pro 185 190 195 ATT CCA TTC TCT GGA TGG CTT AAA TGG TTC CCA GTGTAC ACT TTA 642 Ile Pro Phe Ser Gly Trp Leu Lys Trp Phe Pro Val Tyr ThrLeu 200 205 210 TTC GGT TTC TGT GAT GGA TCT CAC TTC TGG CCA TAC TCT TCACTT 687 Phe Gly Phe Cys Asp Gly Ser His Phe Trp Pro Tyr Ser Ser Leu 215220 225 TTT GTT CGT AAC TCT GAC CGT GTT CAA TGT GTA ATC TCT GGA ATC 732Phe Val Arg Asn Ser Asp Arg Val Gln Cys Val Ile Ser Gly Ile 230 235 240TGT TGC TGT GTG TGT GCA TAT ATT GCT CTA ACA ATT GCT GGA TCA 777 Cys CysCys Val Cys Ala Tyr Ile Ala Leu Thr Ile Ala Gly Ser 245 250 255 TAT TCCAAT TGG TTC TGG TAC TAT TGG GTT CCA CTT TCT TTC TTC 822 Tyr Ser Asn TrpPhe Trp Tyr Tyr Trp Val Pro Leu Ser Phe Phe 260 265 270 GGA TTG ATG CTCGTC ATT GTT ACC TAT TTG CAA CAT GTC GAT GAT 867 Gly Leu Met Leu Val IleVal Thr Tyr Leu Gln His Val Asp Asp 275 280 285 GTC GCT GAG GTG TAC GAGGCT GAT GAA TGG AGC TTC GTC CGT GGA 912 Val Ala Glu Val Tyr Glu Ala AspGlu Trp Ser Phe Val Arg Gly 290 295 300 CAA ACC CAA ACC ATC GAT CGT TACTAT GGA CTC GGA TTG GAC ACA 957 Gln Thr Gln Thr Ile Asp Arg Tyr Tyr GlyLeu Gly Leu Asp Thr 305 310 315 ACG ATG CAC CAT ATC ACA GAC GGA CAC GTTGCC CAT CAC TTC TTC 1002 Thr Met His His Ile Thr Asp Gly His Val Ala HisHis Phe Phe 320 325 330 AAC AAA ATC CCA CAT TAC CAT CTC ATC GAA GCA ACCGAA GGT GTC 1047 Asn Lys Ile Pro His Tyr His Leu Ile Glu Ala Thr Glu GlyVal 335 340 345 AAA AAG GTC TTG GAG CCG TTG TCC GAC ACC CAA TAC GGG TACAAA 1092 Lys Lys Val Leu Glu Pro Leu Ser Asp Thr Gln Tyr Gly Tyr Lys 350355 360 TCT CAA GTG AAC TAC GAT TTC TTT GCC CGT TTC CTG TGG TTC AAC 1137Ser Gln Val Asn Tyr Asp Phe Phe Ala Arg Phe Leu Trp Phe Asn 365 370 375TAC AAG CTC GAC TAT CTC GTT CAC AAG ACC GCC GGA ATC ATG CAA 1182 Tyr LysLeu Asp Tyr Leu Val His Lys Thr Ala Gly Ile Met Gln 380 385 390 TTC CGAACA ACT CTC GAG GAG AAG GCA AAG GCC AAG TAA 1221 Phe Arg Thr Thr Leu GluGlu Lys Ala Lys Ala Lys 395 400 AAGAATATCC CGTGCCGTTC TAGAGTACAACAACAACTTC TGCGTTTTCA 1271 CCGGTTTTGC TCTAATTGCA ATTTTTCTTT GTTCTATATATATTTTTTTG 1321 CTTTTTAATT TTATTCTCTC TAAAAAACTT CTACTTTTCA GTGCGTTGAA1371 TGCATAAAGC CATAACTCTT 1391 402 amino acid residues amino acidsingle stranded linear 2 Met Val Ala His Ser Ser Glu Gly Leu Ser Ala ThrAla Pro Val 5 10 15 Thr Gly Gly Asp Val Leu Val Asp Ala Arg Ala Ser LeuGlu Glu 20 25 30 Lys Glu Ala Pro Arg Asp Val Asn Ala Asn Thr Lys Gln AlaThr 35 40 45 Thr Glu Glu Pro Arg Ile Gln Leu Pro Thr Val Asp Ala Phe Arg50 55 60 Arg Ala Ile Pro Ala His Cys Phe Glu Arg Asp Leu Val Lys Ser 6570 75 Ile Arg Tyr Leu Val Gln Asp Phe Ala Ala Leu Thr Ile Leu Tyr 80 8590 Phe Ala Leu Pro Ala Phe Glu Tyr Phe Gly Leu Phe Gly Tyr Leu 95 100105 Val Trp Asn Ile Phe Met Gly Val Phe Gly Phe Ala Leu Phe Val 110 115120 Val Gly His Asp Cys Leu His Gly Ser Phe Ser Asp Asn Gln Asn 125 130135 Leu Asn Asp Phe Ile Gly His Ile Ala Phe Ser Pro Leu Phe Ser 140 145150 Pro Tyr Phe Pro Trp Gln Lys Ser His Lys Leu His His Ala Phe 155 160165 Thr Asn His Ile Asp Lys Asp His Gly His Val Trp Ile Gln Asp 170 175180 Lys Asp Trp Glu Ala Met Pro Ser Trp Lys Arg Trp Phe Asn Pro 185 190195 Ile Pro Phe Ser Gly Trp Leu Lys Trp Phe Pro Val Tyr Thr Leu 200 205210 Phe Gly Phe Cys Asp Gly Ser His Phe Trp Pro Tyr Ser Ser Leu 215 220225 Phe Val Arg Asn Ser Asp Arg Val Gln Cys Val Ile Ser Gly Ile 230 235240 Cys Cys Cys Val Cys Ala Tyr Ile Ala Leu Thr Ile Ala Gly Ser 245 250255 Tyr Ser Asn Trp Phe Trp Tyr Tyr Trp Val Pro Leu Ser Phe Phe 260 265270 Gly Leu Met Leu Val Ile Val Thr Tyr Leu Gln His Val Asp Asp 275 280285 Val Ala Glu Val Tyr Glu Ala Asp Glu Trp Ser Phe Val Arg Gly 290 295300 Gln Thr Gln Thr Ile Asp Arg Tyr Tyr Gly Leu Gly Leu Asp Thr 305 310315 Thr Met His His Ile Thr Asp Gly His Val Ala His His Phe Phe 320 325330 Asn Lys Ile Pro His Tyr His Leu Ile Glu Ala Thr Glu Gly Val 335 340345 Lys Lys Val Leu Glu Pro Leu Ser Asp Thr Gln Tyr Gly Tyr Lys 350 355360 Ser Gln Val Asn Tyr Asp Phe Phe Ala Arg Phe Leu Trp Phe Asn 365 370375 Tyr Lys Leu Asp Tyr Leu Val His Lys Thr Ala Gly Ile Met Gln 380 385390 Phe Arg Thr Thr Leu Glu Glu Lys Ala Lys Ala Lys 395 400

what is claimed is:
 1. A cell comprising a recombinant FAT-1 polypeptide that desaturates an ω-6 fatty acid to a corresponding ω-3 fatty acid.
 2. The cell of claim 1 wherein the ω-6 fatty acid has a carbon chain of at least 18 carbons.
 3. The cell of claim 2 wherein the ω-6 fatty acid is a 20-carbon ω-6 fatty acid or a 22-carbon ω-6 fatty acid.
 4. The cell of claim 1 wherein the ω-6 fatty acid has a double bond at one or more positions selected from the group consisting of Δ4, Δ5, Δ6, Δ7, and Δ8.
 5. The cell of claim 1 having a proportion of ω-3 fatty acids that is at least 10% greater than an otherwise similar cell lacking the recombinant FAT-1 polypeptide.
 6. The cell of claim 5 having a proportion of ω-3 fatty acids that is at least 20% greater than an otherwise similar cell lacking the recombinant FAT-1 polypeptide.
 7. The cell of claim 6 having a proportion of ω-3 fatty acids that is at least 50% greater than an otherwise similar cell lacking the recombinant FAT-1 polypeptide.
 8. The cell of claim 1 wherein the ω-6 fatty acid has a carbon chain of at least 20 carbons and at least 25% of the ω-6 fatty acid is desaturated to the corresponding ω-3 fatty acid.
 9. The cell of claim 1 wherein the recombinant FAT-1 polypeptide has at least 60% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 10. The cell of claim 1 wherein the recombinant FAT-1 polypeptide has at least 70% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 11. The cell of claim 10 wherein the recombinant FAT-1 polypeptide has at least 80% amino acid sequence a identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 12. The cell of claim 10 wherein the recombinant FAT-1 polypeptide has at least 90% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 13. The cell of claim 1 wherein the recombinant FAT-1 polypeptide has only conservative amino acid substitutions to the FAT-1 polypeptide of SEQ ID NO:
 1. 14. The cell of claim 1 wherein the recombinant FAT-1 polypeptide has 100% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 15. The cell of claim 1 wherein the recombinant FAT-1 polypeptide is encoded by a polynucleotide that comprises a sequence having at least 70% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 16. The cell of claim 15 wherein the polynucleotide comprises a sequence having at least 80% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 17. The cell of claim 15 wherein the polynucleotide comprises a sequence having at least 90% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 18. The cell of claim 15 wherein the polynucleotide comprises a sequence having 100% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 19. The cell of claim 1 wherein the polypeptide is encoded by a polynucleotide comprising a full-length native fat-1 protein-coding region.
 20. A cell of claim 1 of an organism selected from the group consisting of a bacterium, a cyanobacterium, a phytoplankton, an alga, a fungus, a plant, and an animal.
 21. A lipid from the cell of claim
 1. 22. The lipid of claim 21 wherein at least 25% of an ω-6 fatty acid of the cell having a carbon chain of at least 20 carbons has been converted to a corresponding ω-3 fatty acid by the FAT-1 polypeptide.
 23. A transgenic plant comprising a fat-1 polynucleotide that is expressible in at least a part of the plant.
 24. The transgenic plant of claim 23 wherein the fat-1 polynucleotide is expressible in a seed of the plant.
 25. A seed of the transgenic plant of claim
 23. 26. A lipid from the transgenic plant of claim 23 that has a higher proportion of ω-3 fatty acids than a control lipid obtained from an otherwise similar plant lacking the fat-1 polynucleotide.
 27. A method of desaturating an ω-6 fatty acid to a corresponding ω-3 fatty acid comprising the steps of: (a) providing a cell that comprises a recombinant FAT-1 polypeptide; and (b) growing the cell under conditions under which the FAT-1 polypeptide desaturates an ω-6 fatty acid to produce a corresponding ω-3 fatty acid.
 28. The method of claim 27 wherein the ω-6 fatty acid is an ω-6 fatty acid having a carbon chain of at least 18 carbons.
 29. The method of claim 28 wherein the ω-6 fatty acid is a 20-carbon ω-6 fatty acid or a 22-carbon ω-6 fatty acid.
 30. The method of claim 27 wherein the ω-6 fatty acid comprises a double bond at one or more positions selected from the group consisting of Δ4, Δ5, Δ6, Δ7, and Δ8.
 31. The method of claim 27 wherein the recombinant polypeptide has at least 60% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 32. The method of claim 27 wherein the recombinant polypeptide has at least 70% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 33. The method of claim 32 wherein the recombinant polypeptide has at least 80% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 34. The method of claim 32 wherein the recombinant polypeptide has at least 90% amino acid sequence identity with the FAT-1 polypeptide of SEQ ID NO:
 1. 35. The method of claim 32 wherein the recombinant polypeptide has only conservative amino acid substitutions to the FAT-1 polypeptide of SEQ ID NO:
 1. 36. The method of claim 32 wherein the recombinant polypeptide has 100% amino acid, sequence identity with the FAT-1 polypeptide of SEQ:ID NO:
 1. 37. The method of claim 27 wherein the FAT-1 polypeptide is encoded by a recombinant polynucleotide that comprises a sequence having at least 70% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 38. The method of claim 37 wherein the polynucleotide comprises a sequence having at least 80% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 39. The method of claim 37 wherein the polynucleotide comprises a sequence having at least 90% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 40. The method of claim 37 wherein the polynucleotide comprises a sequence having 100% nucleotide sequence identity with the fat-1 polynucleotide sequence of SEQ ID NO:
 1. 41. The method of claim 27 wherein the polypeptide is encoded by a polynucleotide that comprises a full-length native fat-1 protein-coding region.
 42. A method of claim 27 wherein the cell is a cell of an organism selected from the group consisting of a bacterium, a cyanobacterium, a phytoplankton, an alga, a fungus, a plant, and an animal.
 43. The method of claim 42 wherein the cell is a plant cell.
 44. The method of claim 42 wherein the cell is a yeast cell.
 45. A method of producing a lipid comprising an ω-3 fatty acid comprising the steps of: (a) providing a lipid that comprises an ω-6 fatty acid; and (b) desaturating at least some of the ω-6 fatty acid to a corresponding ω-3 fatty acid with a recombinant FAT-1 polypeptide.
 46. The method of claim 45 wherein the lipid comprises an ω-6 fatty acid having a carbon chain of at least 20 carbons, the method comprising desaturating at least 25% of the ω-6 fatty acid to the corresponding ω-3 fatty acid.
 47. The method of claim 45 comprising the step of expressing a recombinant fat-1 polynucleotide in a cell comprising the lipid, thereby producing the recombinant FAT-1 polypeptide in the cell. 